We propose to develop a system for depth-resolved optical imaging of both voltage sensitive dyes (VSDs) and cortical hemodynamics, to enable study of three-dimensional (3D) neurovascular coupling in-vivo (rats). The relationship between neuronal activation and the corresponding hemodynamic response is of fundamental importance for understanding the mechanisms of functional activation, and particularly relevant to interpretation of functional magnetic resonance imaging (fMRI). Once introduced into the cortex, VSDs change their fluorescence proportionally to membrane potential, thereby indicating changes in neuronal activity. We have already developed a system for 3D optical imaging of oxy and deoxy-hemoglobin changes in rat cortex through thinned skull, called Laminar Optical Tomography (LOT). We are proposing to advance LOT.s hardware and algorithms to allow concurrent 3D imaging of rapid, small VSD fluorescence changes in addition to slower hemodynamic absorption changes. The LOT system is similar to a confocal microscope, but rather than varying focal depth, it detects multiply scattered light, which can be used to reconstruct images of structures to depths of >2mm with 100-200 micron resolution. VSD imaging to date has utilized 2D camera images of the cortex, which are very superficially weighted and provide no depth-resolution. Our motivation to simultaneously image VSDs and hemodynamics in 3D is twofold: 1) We hypothesize that to properly quantify the relationship between neural activity and hemodynamics, the two measures must be spatially co-localized in 3D: The depth-sensitivities of 2D fluorescence and absorption images are very different, and so their 2D pixels do not represent the same 3D locations in the cortex. 2) Electrophysiology has demonstrated that neuronal activity is layer-specific. A non-invasive way to study the 3D dynamics of neuronal activation as it moves and spreads between cortical layers would provide a completely new way to study cortical functional activity in-vivo. We propose to develop Fluorescent-LOT (PLOT) and then perform preliminary system testing using rats undergoing somatosensory stimulus. Improved understanding of the correlation between neuronal activity and fMRI signals is of prime importance to human brain imaging. The effects of abnormal pathologies on neurovascular coupling could provide new insights for treatment and prevention. The new system could also find applications in ocular, dermal, endoscopic and tumor imaging. [unreadable] [unreadable]